Safety assessment, radioiodination and preclinical evaluation of antinuclear antibody as novel medication for prostate cancer in mouse xenograft model

This study aims to provide in vitro and in vivo data to support the utilization of antinuclear antibodies (ANAs) as novel tools for the diagnosis and treatment of prostate cancers. The hematological, biochemical, and histological toxicities of ANAs were assessed at the doses of 5 and 50 μg per mouse. Radiolabeling study was then conducted with ANA and 131I using the chloramine T method, and the biodistribution and treatment efficacy were subsequently investigated in a PC3 xenograft model. No changes in clinical behavior or signs of intoxication, necrosis, or malignancy were observed in ANA-treated mice. 131I-ANA was obtained in very high yield and radiochemical purity, at 94.97 ± 0.98% and 98.56 ± 0.29%, respectively. They achieved immunoreactivity fraction of 0.841 ± 0.17% with PC-3 cells. Levels of radiolabeled ANAs were 1.15–10.14 times higher in tumor tissues than in other examined organs at 24 h post-injection. The tumor growth inhibition rates were 28.33 ± 5.01% in PC3 xenografts mice treated with 131I-ANAs compared with controls and a nearly twofold improvement in median survival was observed. These results demonstrate that radioimmunotherapy of radiolabeled natural ANAs may be an effective treatment for prostate tumors.

www.nature.com/scientificreports/cells are present.Furthermore, in malignant tumors, more than 50% of tumor cell progeny quickly degenerate or even undergo necrosis.Therefore, the membrane permeability of degenerating tumor cells increases, allowing natural antinuclear antibodies to easily bind to nuclear antigens in these abnormal cells 1 .
Relying on this mechanism, many test results have been reported regarding the recombination of monoclonal anti-DNA antibodies, such as the 3E10, 3D8, 5C6, 2C10, H241 and G2-6 antibodies, among which the 3E10 mAb has been tested in patients, and its safety and efficacy have been proven in the clinic 1,7 .Nevertheless, monoclonal anti-DNA antibodies may not represent pathological antibodies in cancer.In addition, such antibodies are expensive, which makes it difficult to use these antibodies to treat patients 12 .However, ANAs are inexpensive, are readily available, have minimal side effects and are effective against a broad spectrum of tumors 4,5 .Many studies on autoantibodies have shown that combination with radioisotopes or chemicals may increase tumor cell sensitivity to these autoantibodies 13 .For example, tumor hypoxia and necrosis can be therapeutically targeted with α-radioconjugates and 131 I-labeled tumor necrosis therapy (Cotara) in patients with developed lung cancer 14,15 .Therefore, this created an opportunity to promote in vitro and in vivo studies on ANAs and produce new radiopharmaceuticals with potential for use in radioimmunotherapy for treating various tumor types.
Prostate cancer is one of the most prevalent cancers in men, and it has a high mortality rate.According to Globocan 2020, prostate cancer is the second most common cancer, accounting for 14.1% of all new cancer cases and ranking sixth in mortality.The treatment of prostate cancer has become one of the greatest challenges in modern medicine 16 .A novel radioactive therapy called 68 Ga/ 177 Lu-PSMA is currently being used to treat prostate cancer 17 .Besides, the development of radiopharmaceutical therapy for prostate cancer base on the labeled antibodies such as 225 Ac-PSMA-617, 177 Lu/ 225 Ac-J591, 255 Ac/ 90 Y-hu5A10, 177 Lu-SC16, 225 Ac-YS5 for preclinical application on prostate cancer were evaluated 17 , but it is not currently available in Vietnam because imports are expensive.As a result, a simpler the study's model for prostate cancer treatment was chosen since it was based on ingredients that were easy to obtain and utilize.Similarly, radioisotope 131 I is a candidate that is suitable for labelling ANAs, and over recent decades, it has been shown to have high clinical safety and gamma 364 keV and beta 606 keV energy 3,18,19 . 131I-ANA conjugates have high potential for use in diagnosing and treating cancer.In this report, the safety of ANAs in tissues was tested.Then, we determined the ability of ANAs to be radiolabeled with the 131 I radioisotope.Finally, the capacity of this labeled conjugate to bind to a prostate cancer cell line, its biodistribution and its efficacy in the treatment of human PC3 tumor-bearing nude mice were evaluated.The schematic illustration of the study was presented in Fig. 1.

Safety profile of ANA injection
Clinical observations: No mortality was observed in any group.All the mice looked healthy, agile, and ate and drank normally while being fed according to the supplier's instructions.There were no unusual changes in appearance, behavior or locomotor activities and no clinical signs of toxicity.
Body weight measurement: All the mice in Group I gained weight (P < 0.05) on Days 1 and 3, and all the mice in Group II gained weight (P < 0.05) on Days 1 and 30.There were also no significant differences (P > 0.05) in the change in body weight between the treatment groups and control groups (Table 1).
Hematological findings: In general, the hematological parameters of the 5 μg ANA-injected groups were not significantly different from those of the control groups (P > 0.05).However, an opposite phenomenon was observed in the 50 µg ANA-injected groups, as red blood cell and platelet numbers were consistently lower than those in the control groups (Table 2).
Biochemical findings: The injection of ANAs had no significant effect on SGPT (P > 0.05), whereas increases in SGOT were observed in mice that were injected with 50 μg ANAs on Day 3 p.i. (P < 0.05) (Table ).However, the level of SGOT returned to the normal range on Day 30 p.i., and this level was not significantly different from that in the control group (P > 0.05) (Table 2).www.nature.com/scientificreports/Histopathological findings: Spleen, kidney, heart and lung tissues from every group exhibited no significant abnormalities.No signs of necrosis or malignancy were evident from the microscopic views of these organs.However, noticeable histopathological changes in liver tissues were observed (Fig. 2).In particular, mild reactive inflammation in Group IIb (injected with 5 μg ANAs, Day 30), a few cave-degraded hepatocytes in Group Ic (injected with 50 μg ANAs, Day 3) and additional signs of bile accumulation in Group IIc (injected with 50 μg ANAs, Day 30) were observed, as opposed to the minimal histopathological changes that were in Groups Ia, IIa (normal saline control group) and Ib (injected with 5 μg ANAs, Day 3).There was light venous congestion in the tissues on Day 3, which disappeared on Day 30.
Three days after 5 µg of ANAs were injected, the tissues showed signs of inflammation and congestion, with the liver tissue showing 20% congestion and 20% inflammation.The spleen and heart exhibited 20% congested, although the lungs and kidneys exhibited only 10% congestion.The majority of the tissues returned to their normal functional states during the course of the 30-day follow-up, while the congestion of the livers barely Table 1.Body weight measurements and body weight gain (grams) of mice in all groups (n = 10).Note that in each group, there were no significant differences in both baseline weight and weight gain between types of treatment (P < 0.05).

Parameters
Group I (3 days) Group II (30 days)   www.nature.com/scientificreports/decreased (from 20 to 10%).In addition, congestion and cave-degraded hepatocytes were observed in organ tissues three days after injection.Over 30 days of continued observation, the congestion of liver tissues remained unchanged.However, congestion in other organs decreased.

Preparation and characterization of 131 I-ANAs
The ANA's radiolabeling efficiency affected factor included chloramine T, pH, ANA content and incubation time and the results of determination are presented in the Supplementary Fig. S1.Hence, it was chosen in the labeling with 131 I to generate a 131 I-ANAs.The yield of radiolabeling was 94.97 ± 0.98% (n = 6), as shown by the representative data in Fig. 3a.The paper electrophoresis results showed a labeled antibody peak with R f ~ 0.1-0.3 at the origin.Under level labeling reaction conditions of 341 ± 33.9 MBq (12.09 ± 1.2 nmol) 131 I and 1.0 mg (6.6 nmol) ANAs, 1:33 molar ratio of ANAs and chloramine T (chT) and the free 131 I was removed by gel chromatography, as shown in Fig. 3b, in fractions 9 and 10.The concentration of collected 131 I-ANAs was 108 ± 11.5 MBq/mL according to a dose calibrator (Capintec ISOMED 2000, USA), pH 7.4.The radiochemical purity was 98.56 ± 0.29% (Fig. 3c).The 131 I-ANAs in Fig. 3d were collected and had a specific activity of 335.88 ± 36.58MBq/mg, and the average number of iodine atoms per ANA molecule was estimated to be 1.74 ± 0.19 (Table 3).The radioconjugates were stable after 16 days in 0.9% NaCl in 0.05 M PBS at 4 °C and -20 °C, and they remained stable for 9 days in 0.05% human serum at 37 °C; these samples exhibited 96.3 ± 0.31% radiochemical purity (Fig. 3e and 3f).

Immunoreactivity and saturation binding assay of 131 I-ANAs
The immunoreactivity fraction (r) of the 131 I-ANAs was 0.841 ± 0.17% (best fit value ± standard error), which is shown in Fig. 4a.The saturation binding assay revealed a Bmax value of 4.48 ± 0.57 amol/cell (best-fit value ± standard error of the mean), which is approximately 2.7 × 10 6 atoms/cell.Additionally, the affinity of 131 I-ANAs for PC3 cells was demonstrated, and the Kd value was 16.15 ± 4.3 nmol/L (best-fit value ± standard error).The nonspecific binding was insignificant (< 3%).The results of the saturation binding assay are shown in Fig. 4b.In the figure, the horizontal axis represents the increasing concentration of 131 I-ANAs (nmol), and the vertical axis represents the number of antibody binding sites per cell (amol/cell-specific binding).The results were analyzed by nonlinear regression using GraphPad Prism 8 software.When the graph reaches the maximum value, this indicates saturation.The nonspecific binding fraction was less than 3%, and the difference was significant (P < 0.05%).

Cell apoptosis analysis
The percentage of apoptotic cells in the 131 I-ANA-treated groups was significantly higher than that in the control groups (P < 0.01).After 48 h of the experiment, early apoptosis was induced in 19.43 ± 2.96% of PC3 cells that were treated with 131 I-ANAs, and this proportion was 4.28 times higher than that in the control group (4.53 ± 1.1%).At 72 h, 131 I-ANAs caused 29.77 ± 4.95% of PC3 cells to undergo early apoptosis, increasing the proportion of apoptotic cells by approximately 5 times compared with that in the control group (6.0 ± 1.2%).(Fig. 4c and 4d).

Pharmacokinetics, biodistribution and treatment efficacy of 131 I-ANAs
The biodistribution of 131 I-ANAs at 6, 24, 48, 72, and 168 h p.i. is shown in Table 4 and Fig. 5a.In the first 48 h p.i., 131 I-ANAs were generally distributed to the PC3 tumor as much as to other organs, with marginal differences at 24 h and 48 h p.i. (P < 0.05).Then, 131 I-ANAs were predominantly retained in tumor tissues 72 h after administration (P < 0.001), with the only exception being liver tissue.Although the uptake of 131 I-ANAs by the liver was even higher than that by the tumor in the first 72 h p.i., the levels gradually decreased from their peak of 8.47 ± 1.88% ID/g at 6 h p.i. to 4.22 ± 1.5% ID/g at 168 h p.i..Moreover, 131 I-ANAs accumulated and remained stable at the PC3 tumor site, with a fluctuation within 1.38% ID/g from its highest value of 5.83 ± 3.24% ID/g at 48 h p.i.Other noncancerous organs also exhibited a pattern of decreasing uptake after the levels peaked in the first 24 h, but these other organs exhibited faster rates of decline than the liver.The levels of 131 I-ANAs in tumor tissues were 1.15 to 10.14 times higher than those in other examined organs 24 h p.i., except for the levels in liver tissues.The half-life of labeled ANAs in the blood was 31.85 h (R square = 0.7163, Fig. 5b).The body weight examination was exhibited no significant differences compared to mice treated with 0.9% NaCl, ANAs and 131 I-ANAs (P > 0.05) which are shown in Supplementary information, Fig. S2.The initial tumor sizes in the 0.9% NaCl, ANAs and labeled ANAs treatment groups were 441 ± 192, 495 ± 254 and 443 ± 211 mm 3 , respectively.After 45 days, the tumor sizes were 5982 ± 384, 4925 ± 826 and 3949 ± 917 mm 3 , respectively.Mice that were treated with ANAs or 131 I-ANAs had significantly smaller tumor volumes than control mice (****P < 0.0001; two-way ANOVA with multiple comparisons), and mice that were treated with ANAs had significantly smaller tumor volumes than 131 I-ANA-treated mice (***P = 0.0006; two-way ANOVA with multiple comparisons).The tumor growth inhibition rates of the 131 I-ANA and ANA groups compared with the 0.9% NaCl group were 28.33 ± 5.01% and 17.41 ± 5.18%, respectively (Fig. 5c).The tumor growth inhibition rates of the 131 I-ANA compared with the ANA group were 13.21 ± 6.79%.Kaplan-Meier survival analysis showed that treatment with 131 I-ANAs or ANAs increased the median survival of mice with PC3 xenografts by 1.83-fold or 1.43-fold, respectively, compared with treatment with 0.9% NaCl (P = 0.0001; chi-square 14.44; or P = 0.209; chi-square 1.57, log-rank (Mantel-Cox) test) (Fig. 5d).The survival of mice was also monitored until 50% of the control group had died.On Day 45, the mortality rates in the groups that were treated with 131 I-ANAs, ANAs and 0.9% NaCl were 20, 40 and 50%, respectively.Interestingly, 100% of mice in the 0.9% NaCl and ANA groups had died by Day 60 and Day 75, while 20% of the mice that were treated with 131 I-ANAs survived.Histopathological analysis of liver and kidney tissues showed hemorrhagic zones, mild degeneration, and dilated portal vein disorder, but no human prostate

Discussion
In this study, we demonstrate that human ANAs could be labeled with 131 I with high efficiency.The optimal research conditions led to 131 I-ANA conjugation with a high labelling efficiency of 94.97 ± 0.98%, which is consistent with published studies.Furthermore, the successful binding of the ANAs to the tumor cell line indicates its potential for use in clinical applications.In addition, we also determined, including by histopathology, that concentrations of ANAs between 5 and 50 µg are safe in experimental mice.The first reason we chose ANAs in this study is because natural ANAs exists inside the body, even in elderly individuals, which proves that ANAs are safe for living creatures.Second, ANAs can destroy cancer cells in the necrotic area 1,5,8 .Furthermore, several authors have shown that SLE patients are protected against several cancers, such as lung, prostate, and breast cancer 7,23 .Recently, two-thirds of diseases have been shown to be related to targets within nucleus, especially dsDNA and nucleosomes 1,4 .In addition, ANAs are easily isolated from SLE patients and are used as a therapeutic agent 1,4,5 .Regarding a hypothetical mechanism, ANAs bind to nucleosomes that are released from apoptotic tumor cells, circulate inside and outside the tumor and kill tumors via antitumor mechanisms, such as ADCC/ immune effector cells 5 .These mechanisms have enabled the development of novel methods for the diagnosis and treatment of several cancers, as proposed in previous studies 30 .In terms to the ANA dose that was used in the experimental mice, we administered between 5 µg and 50 µg ANAs per mouse, and this dose was based on a previous similar study that used humanized monoclonal antibodies, such as rituximab, cetuximab, and nimotuzumab.The safety results showed the weight, hematological parameters and biochemical parameters of the mice that were treated with 5-50 µg ANA were within normal ranges.Studies have reported that the dose for Bexxar antibody treatment is 450 mg per patient, that for rituximab treatment is 225 mg per patient 31 , and that for nimotuzumab treatment is 200-400 mg per patient (average 2.8-8.5 mg/kg); nevertheless, the dose for treatment with radiolabeled antibodies was approximately 0.36-1.0mg/kg.Therefore, a dose of 1 mg/kg was chosen for use in the experiments 32,33 .When injecting ANA, an antibody highly presented in SLE patients, into healthy individuals, concerns about recreating SLE symptoms may be raised.Although ANAs present at high titer in SLE patients, they can also be found in healthy people, at low ANA titer (< 1:160) 34 , and the understanding of autoimmune physiology revealed that auto-antibody does not warrant the acquisition of SLE.However, ANA titer seems to be related to disease severity: if the ANA titer is high (e.g.1:640, 1:1280, or 1:2560), this indicates a more severe disease.If the ANA titer is low (e.g.1:40, 1:80, or even 1:160), there is often no autoimmune disease 4 .Different tissues from the mice in the different groups showed no significant abnormalities, with only mild reactive inflammation in the liver and kidney.The injected ANAs did not result in the destruction of normal tissues.However, additional studies are required to determine the safety of 131 I-ANAs with respect to other tissues.We demonstrated that ANAs can be labeled with the radioisotope 131 I, which enhanced in in vitro and in vivo ANA studies.Additionally, 131 I was selected because it is suitable for targeted radioimmunotherapy in the treatment of small tumors, such as prostate cancer, and treatment can be followed with SPECT imaging.Second, 131 I Table 4. Biodistribution of 131 I-ANA in different tissues of PC3 xenograft mice, N = 5 per condition.Within the same column, asterisk (*) means that at the same time point post-injection, the levels of 131 I-ANA in organs differ significantly from those in the tumors (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA tests with Dunnett's T3 multiple comparisons).Test details on 168 h: Tumor with blood (p = 0.076, t = 5.9) Tumor with heart (p = 0.012, t = 4.7), Tumor with lung (p = 0.0043, t = 7.5), Tumor with kidney (p = 0.006, t = 8.5), Tumor with intestine (p = 0.0031, t = 8.1).T/B, tumor to blood ratio; T/M, tumor to muscle ratio; T/L, tumor to liver ratio; T/K, tumor to kidney ratio; T/Th, tumor to thyroid ratio.www.nature.com/scientificreports/www.nature.com/scientificreports/emits both γ-and β-rays.Furthermore, 131 I is a well-known radioisotope and is frequently used for treating thyroid cancer.We used a traditional radiolabeling modality that is similar to that used for labeling antibodies with 131 I, such as 131 I-tositumomab, 131 I-rituximab, 131 I-nimotuzumab, 131 I-omotuzumab and 131 I-bevacizumab; the results showed high labelling efficiency (> 94%), similar to a previous study that reported > 91% efficiency, high radiochemical purity (> 98%) and in vitro stability (> 96%) for 16 days 22,23,26 .The immunoreactivity study results showed that radiolabeled ANAs did not significantly affect immunoreactivity or affinity.The immunoreactivity fraction r of 131 I-ANA was 0.841 ± 0.17%, which was similar to previous reports on radioimmunoconjugates that reported ~ 0.8 for 31 I-nimotuzumab, > 80% for 131 I-bevacizumab 26,35 or 83% for 131 I-omburtamab (8H9) 36 .The binding capacity of the 131 I-ANA complex to PC3 cancer cells was measured; the K d value was approximately 16.15 ± 4.3 nmol/L (10 -9 M), and the number of sites on the cytoplasmic antigen calculated from the B max value was 2.7 × 10 6 .The 10 -9 M data confirm antibody binding affinity after radiolabeling 3 .These results are similar to those reported by studies on 177 Lu-nimotuzumab (K d values were 28.6 ± 3.9 nmol/L and 32.1 ± 5.5 nmol/L, and binding sites per cell were 1.7 × 10 6 and 7.1 × 10 5 for A431 and SNU-C2B cells, respectively 36 ), 131 I-nimotuzumab (Kd value ~ 24.7 nmol/L and 19.2 × 10 5 binding sites per cell with Hep-2 cells) 22 , and 99m Tc-fanolesomab (K d ~ 1.6 × 10 -11 M and 5.1 × 10 5 binding sites per human neutrophil 37,38 ).The results showed that the specificity and affinity of human ANAs for tumor nuclear antigens were higher than or equivalent to those of specific monoclonal antibodies on the tumor cell membrane.After incubating PC3 cells with 131 I-ANAs for 48 h and 72 h, the PC3 cells were observed, and the results revealed an effect of 131 I-ANAs on inhibiting PC3 cell growth.These results are similar to results from studies on the tumor necrosis-inducing effects of the 131 I-factor-related apoptosis-inducing ligand (TRAIL-I-131) to the A549 and H358 cells 39 .Consequently, the data related to the 131 I-ANA biodistribution in PC3 tumor-bearing mice indicated high radioactivity concentrations in the liver, kidney, blood, and lungs and retention in the heart and blood after 168 h of monitoring.It would be reasonable that the biological half-life of ANAs was 3-4 weeks.After one week of follow-up, the radioactivity concentrations in cancer tissues were 10 times higher than those in kidney tissues, 5 times higher than those in muscle tissues, and 3 times higher in blood.Perhaps one of the reasons was the increased levels of DNA in the necrotic regions of the tumor, indicating the possible use of 131 I-ANAs in therapeutic trials and follow-up scintigraphy.
The results were relatively similar to those from other similar studies on the biodistribution of radiolabeled antibodies in xenografts.Additionally, these findings were consistent with the 20 μg 64 Cu-cetuximab per mouse in a study by Yukie et al. 40 .We demonstrated that labeled ANAs exert antitumor effects in PC3 tumors with a large sample size (n = 30 mice) and repeated injection.The in vivo data on the tumor growth inhibitor rate and survival were significant, and 131 I-ANA injection resulted in a 1.83-fold increase in the median survival of mice compared to the control.

Conclusions
In summary, we have shown that natural antibodies isolated from SLE patients are safe to utilize in experimental mouse models.Concurrently, ANAs were successfully radiolabeled with 131 I via high-efficiency radioiodinated conjugation, and these labeled antibodies showed high radiochemical purity as well as stability in human serum.
The remarkable results of in vitro and in vivo 131 I-ANA characterization in prostate cancer cell lines and PC3 xenograft mouse models demonstrated that this radioimmunoconjugate is a potential agent that could be suitable for further therapeutic study for treating not only prostate cancer but also various other types of cancer.Further studies should concentrate on the dose so that radioconjugated ANAs can be used clinically and explored in other solid tumor models to exploit their therapeutic potential.

Ethics declarations
The study is reported in accordance with ARRIVE guidelines.All the animal care and experimental protocols were approved by the animal ethics committee of the Vietnam Military Medical University and followed the guidelines of the Animals (072/13).Throughout the experimental period, the mice were raised in a clean room and provided with filtered air, food and drink according to the manual of the mouse provider.

ANA safety studies
Sixty 8-week-old male Swiss mice were obtained from Nha Trang Pasteur Institute (Nha Trang, Vietnam), randomly divided into 2 groups of equal size, and investigated for 3 or 30 days.In each group, the mice were divided into 3 subgroups of 10 mice each.The mice in subgroup a were injected with normal saline and served as control groups, while 5 μg or 50 μg ANAs (Probactive Biotech, Inc. CA, USA) 8 were injected into the mice in subgroups.The clinical behavior of all the mice was observed twice daily.For the mice in Group I, body weight was measured on Days 1 and 3 postinjection (p.i.).Blood samples were harvested on Day 3 to count RBCs, WBCs, and PLTs using a hematological analyzer (XN-1000, Sysmex, Japan), and quantification of SGOT and SGPT was performed using a biochemistry analyzer (AU 680, Beckman Coulter, Japan).Similar investigations were performed on the mice in Group II, but the timing of body weight measurement (on Days 1 and 30) and blood sample analysis (on Day 30) were altered.At the end of the experimental period, the mice were euthanized with ketamine and xylazine.Their spleens, kidneys, hearts, lungs and livers were excised, fixed in 10% neutral buffered formalin for 24 h before undergoing a series of histopathological processes for sample preparation 20 , hematoxylin and eosin staining 21 22,23 base on results on reaction optimization.Briefly, 100 μL of 0.5 M PBS, pH 7.4, 606 μL (1.0 mg) of ANAs, 50 μL (296-370 MBq) of Na 131 I and 50 μL (50 μg) of Chloramine T (Sigma) were consecutively added to the reaction vial, lightly mixed and incubated for 5 min at room temperature.To stop the reaction, 75 μL (150 μg) of sodium metabisulfite was added and gently mixed.The process of labeled ANA purification was performed on a gel filtration pD10 column (Sephadex G25, GE Healthcare Buckinghamshire, UK), and the samples were preequilibrated and eluted with 0.05 M PBS, pH 7.4, supplemented with 1.0% human serum (Sigma-Aldrich).Fractions 4, 5 and 6 exhibited the highest radioactivity, and they were pooled and then subjected to sterile filtration with a 0.2 µm filter (Sartorius, Goettingen, Germany).The specific activity of 131 I-ANA in MBq/mg was estimated by calculating the radioactivity of 131 I and the mass of ANAs after UV spectrophotometry at 280 nm.The number of 131 I atoms that bound to each ANA molecule was calculated by multiplying the molar ratio of iodine per molecule of ANA by the labeling efficacy as previously described 22,23 .In the experiment, we used 1.0 mg (6.6 nM) of ANA and 370 MBq (13.12 nM) of 131 I based on the specific activity of 131 I, which is 222 GBq/mg, and the molecular weight of the ANAs, which is 150 kDa.The labeling efficacy and radiochemical purity were determined by paper electrophoresis (0.025 M PBS, pH 7.5, 300 V, 60 min, Whatman 1 paper strips) 24 and confirmed by Tec-Control Chromatography strips (Biodex Medical Systems, Inc., NY) 25 27 .In short, a fixed concentration of 131 I-ANAs (25 ng/mL (20.3 KBq/mL) were incubated for 2 h at 37 °C with increasing concentrations of PC3 cells (0.2 to 9 million cells/mL).To determine nonspecific binding, 100-fold excess cold nonradiolabeled ANAs were used to saturate specific binding sites before adding 131 I-ANA.After incubation, the radioactivity in the tubes was measured, the cells were transferred to the wells in a Millipore tray (Filter Plate 96-Well, Millipore), the Millipore filter system was installed, the pump was turned on to drain the solution.Then, the cells were washed with phosphate buffer 4 times, the membrane filter containing cells was collected, and the radioactivity was measured with a gamma counter (Caprac 13, Capintec) together with that of the total tube.A double inverse plot of total applied/specific binding activities (after nonspecific binding activities were subtracted) as a function of 1/cell concentration yielded a straight line.

Saturation binding assay
A fixed number of PC3 cells (2.0 × 10 5 cells) were incubated with or without 100-fold excess cold nonradiolabeled ANAs to block nonspecific binding; then, the cells were incubated for 3 h at 37 °C with a doubling concentration of 131 I-ANA (1 nM to 50 nM) 28 .Then, the cells were washed and collected through a Millipore membrane, and radioactivity was measured.The binding affinity (Kd) of the 131 I-ANAs and the maximum binding capacity (Bmax) were determined by plotting the concentration of 131 I-ANAs bound to the cells (amol/cell) as a function of the 131 I-ANA concentration and using nonlinear regression analysis.

Cell apoptosis analysis
PC3 cells were subcultured into 4 groups that each contained three 25 cm 2 culture flasks (Corning) with 10 5 cells each.Two groups were continuously cultured in F12K medium with the addition of 10.3 μg (100 μCi/118 μL) of 131 I-ANA, while the other 2 groups were treated with PBS as a control.After 48 h and 72 h, the apoptosisinducing property of 131 I-ANAs was determined by using an Annexin V-PI dead cell apoptosis kit (Invitrogen, Massachusetts, USA) according to the manufacturer's instructions.Briefly, 100 μL of a 10 6 cell/mL suspension was incubated in the dark with 100 μL of Annexin V/PI for 15 min at room temperature.The cells were observed by fluorescence microscopy.Flow cytometry (Beckman Coulter, CA) and FACSDiva Software 6.1.3were used for investigation and data analysis.

Evaluation of biodistribution and treatment efficacy
To establish tumor-bearing mouse models, 10 8 PC3 cells (100 μL) were subcutaneously injected into the right thigh of 8-week-old BALB/c nude mice (Charles River Laboratory, Morocco, USA).The tumor volumes of 75 mice reached ~ 400 mm 3 , which was calculated with the tumor length (L) and width (W) according to the following formula: LW2 × 0.5 22 .In the biodistribution experiment, 25 BALB/c mice with PC3 xenografts were divided into 5 groups of 5 mice each to perform an investigate with 5 time points (6, 24, 48, 72, and 168 h p.i.). 131I-ANAs (15.3 μg; ~ 5.18 MBq/100 μL) were injected into the mice via the tail vein.At each designated time point, the mice in the corresponding group were sacrificed, and their organs of interest and blood samples were collected, weighed, cut and examined to measure residual radioactivity.By comparing the radioactivity of the injected dose (ID) and considering the natural disintegration of 131 I, the biodistribution of 131 I-ANAs was expressed as the percentage of ID per gram of tissue at the time of measurement (% ID/g) 29 .The blood clearance half-life of the radio-conjugates was calculated using one-phase exponential decay (GraphPad Software Inc.).In the treatment experiment, 50 BALB/c mice with PC3 xenografts were divided into 3 groups and given six doses of weekly injections with 100 μl 0.9% NaCl (n = 10), 20 μg ANAs (n = 10) or ~ 7.33 MBq/20 μg 131 I-ANAs (n = 30).
In the 131 I-ANAs group, Lugol was added to the drinking water 2 days before the treatment began and refreshed daily, to block the thyroid from taking up free radioiodine.The tumor size and body weight were measured every 3-4 days.The tumor growth inhibition rate (%) was calculated as follows: 100 − (the mean tumor size of the treatment group/mean tumor size of the control group) × 100 22 .The treatment efficacy in the mouse groups was analyzed using GraphPad Prism 8. Histological examination of the tumor, liver and kidney samples from the 72 and 168 h groups was carried out with hematoxylin and eosin staining and observation under light microscopy.

Statistical analyses
Data are expressed as the means ± SDs/SEM.Statistical analysis was performed with GraphPad Prism 8, and a P value of < 0.05 was considered significant.The normality of the datasets was determined by the Shapiro-Wilk test.Student's t test was used in this research to analyze and estimate the differences in body weight, hematological and biochemical parameters and biodistribution.Alternatively, the Kruskal-Wallis test followed by Dunn's post hoc test was used to analyze nonparametric data.

Figure 1 .
Figure 1.The schematic illustration of study.ANAs Anti nuclear antibodies.

Figure 2 .
Figure 2. Representative images of different tissues from mice in all groups that were stained with hematoxylin/ eosin.No significant abnormalities were evident in any sections.a Group I, observed on Day 3 after injection; Control group (normal saline); 5 μg ANA-treated group (showed signs of inflammation and congestion, with the liver tissue showing congestion in 2/10 mice and inflammation in 2/10 mice).The spleen and heart exhibited congestion in 2/10 mice, and the lungs and kidneys exhibited congestion in 1/10 mice; 50 μg ANA-treated group (showed signs of congestion in 4/10 mice of each group); Group II, observed on Day 30 after injection; Control group (normal saline); 5 μg ANA-treated group (showed signs of inflammation in liver tissue in 1/10 mice); 50 μg ANA-treated group (1/10 mice showed cave-degraded hepatocytes, 1 mouse in each group showed light vein congestion); Ia, IIa: No significant abnormalities were evident in any sections.White arrow: Vein congestion.Gray arrow: Mild reactive inflammation with a cluster of lymphocytes.Black arrow: Cave-degraded hepatocytes.

Figure 3 .
Figure 3. Radiolabeling and quality control of 131 I-ANAs.(a )Efficiency yield of 131 I-ANAs as determined by paper electrophoresis in 0.025 M phosphate buffer and radioautography (Cyclone, PerkinElmer).Chromatograms were analyzed using OptiQuant 5 software. 131I-ANAs stayed at the anode R f = 0.0-0.25, and free 131 I was separated from the reaction mixtures and migrated to the cathode R f = 0.75-0.85.(b) 131 I-ANAs were collected in fraction no.3-6 mL after purification through gel sephadex, and free 131 I was collected in fraction no.8-10 mL.(c) Radiochemical purity of 131 I-ANAs as determined by Tec-Control-Chromatography (TCC 150-771), Biodex.The strip was placed into a vial containing 0.9% saline, and the strip was developed until the solvent front migrated to the solvent front line.The strip was removed, and then the storage phosphor screen was used.Chromatograms were analyzed using OptiQuant 5 software. 131I-ANAs remained at the origin R f = 0.0-0.3, and free 131 I was separated from the reaction mixtures and migrated to the front R f = 0.9-1.0.(d) Hypothetical chemical structures of 131 I-ANA conjugates, 131 I attachment to the ortho site of the phenol ring tyrosine.(e) Stability of 131 I-ANAs in 0.9% NaCl and 0.05 M PBS after cryopreservation and storage 4 °C.All experiments were performed in duplicate and 3 independent times (n = 3).Simple linear regression analysis of the results was performed by GraphPad Prism Software 8. (f) 131 I-ANAs in 0.05% human serum were stable (96.3 ± 0.31% radiochemical purity) after incubation at 37 °C.

Figure 4 .
Figure 4.In vitro characterization of 131 I-ANAs in PC3 cells.(a) Immunoreactivity of 131 I-ANAs against PC3 cells.Duplicates were used to acquire each data point, and the results are shown as the mean ± standard deviation (error bar).Representative results of three independent Lindmo assays to calculate the immunoreactivity (r) of 131 I-ANAs.By using linear regression analysis 3, immunoreactivity was calculated as the inversion of the Y-intercept.(b) 131 I-ANA saturation binding assay; results are representative of three independent assays of 131 I-ANA binding to PC3 cells.The Kd and Bmax values are shown as the bestfit value ± standard error.(c) PC3 cell line after incubation with 131 I-ANAs and observed by fluorescence microscopy, (c)1: control (after culture), (c)2: 48 h, (c)3: 72 h, c4: PC3 cells stained with annexin V (early stage of apoptosis), (c)5: PC3 cells stained with annexin V and PI (late stage of apoptosis), (c)6: PC3 cells stained with PI.(d) Apoptosis analysis: PBS and 131 I-ANA treatments at 48 and 72 h.Flow cytometry and FACSDiva Software 6.1.3were used for investigation and data analysis (n = 3, **P < 0.01; Student's t test).The results are presented as the mean ± standard error.The apoptotic profile was determined as follows: living cells were Annexin V (−) and PI (−), early apoptotic cells were Annexin V ( +) and PI (−), late apoptotic cells were Annexin V ( +) and PI ( +), and dead cells were Annexin V (−) and PI ( +).

Table 2 .
Count of red blood cells (RBC), white blood cells (WBC), platelets (PLT), serum glutamicoxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase in mice of all groups (SGPT).n = 9-10 for each group.( # ) Mouse biochemistry normal ranges are referred from en.wikivet.netreference.Within a column of the same hematological parameters, those marked with an asterisk (*) are different from the corresponding control group (P < 0.05).Within a column of the same biochemical parameter, those

Parameters/References 41 Group I (3 days) Group II (30 days)
and finally examination by light microscopy.To compare the data from repeated weight measurements, a paired t test was utilized.To compare data between 2 groups, Student's t test with Welch's correction or the Mann-Whitney U test for nonparametric data was utilized.To compare data among multiple (> 2) groups, one-way Welch's ANOVA test followed by Dunnett's post hoc test for comparison to a specific group of interest was utilized.Radiolabeling of ANAs (1.65 mg/mL, in phosphate-buffered saline (PBS), pH 7.4) with sodium iodine-131 I (Na 131 I, 222 GBq/mg, 7.4 GBq/mL in 0.05 N NaOH, > 99.9% radionuclide purity, Dalat, Vietnam) was performed with the Chloramine T method 26After iodination, aliquots of 131 I-ANA were stored in 0.9% NaCl in 0.05 M PBS at either 4 °C or -20 °C for 16 days (9.25 MBq, 13.7 μg ANA, 0.5 μL), and in human serum and 0.9% NaCl in 0.05 M PBS at 37 °C for 9 days (12.95 MBq, 19.2 μg, 0.5 μL) for stability studies using paper electrophoresis.The radioactivity in the paper electrophoresis strips was scanned by radioautography (Perkin Elmer Life Science)26.The preparation was performed in 6 batches during the experiment.